The importance of acetylcholinesterase (AChE) lies in its physiological role in neurotransmission, its prominence as a marker for nerve-muscle interactions, and because it is a complex family of oligomeric forms with multiple subcellular locations. Studies of this enzyme will detail how muscle cells regulate the synthesis and assembly of synaptic components in particular, and in broader terms, will provide specific information concerning the mechanisms of regulation and localization of membrane and secreted proteins. The goal of this proposal is to determine the molecular and cellular mechanisms regulating synthesis, assembly, degradation, and localization of the multiple oligomeric forms of AChE in muscle. The overall objective is to determine the relative contributions of the many post-translational events in regulating AChE localization and metabolism. The specific aims are to: 1) determine whether differences in primary structure alone account for the different oligomeric forms and subcellular localizations of AChE; these studies entail isolating isotopically labeled AChE polypeptides from different subcellular compartments and comparing their primary sequences by peptide mapping; 2) determine the structural differences between active and inactive AChE molecules and the cellular mechanisms involved in sorting the two classes; these studies employ a variety of pulse/chase paradigms in conjunction with other biochemical methods to identify modifications, and to determine when and where in the cell these modifications occur; 3) identify the subcellular location of the several AChE forms by indirect immunofluorescence and electron microscopy following differential extractions and enzymatic treatments; 4) study the specific mechanisms of intracellular degradation, assembly, and processing of the AChE polypeptide chains in order to 5) study the role of muscle activity in regulating the synthesis, activation, assembly, transport, and ultimately localization of the multiple AChE forms.